Free flow isoelectric focusing (FF-IEF) is a mode of electrophoresis that occurs in a pH gradient. FF-IEF can continuously separate and concentrate amphoteric molecules on the basis of their isoelectric point (pI). Generally, FF-IEF involves flowing a sample solution containing a mixture of species through a rectangular chamber and applying an electric field to establish a pH gradient across the width of the chamber. The species within the mixture may be separated across the width of the rectangular channel according to the interaction of the different species with the pH gradient.
FF-IEF has been employed on a preparative scale, wherein a typical chamber may have a channel width on the order of at least tens of centimeters. Generally, in such cases, the distance between electrodes on either side of the channel may reduce the effectiveness of the electric field in establishing the pH gradient, and the pH gradient must be artificially established by the introduction of pH buffers into the channel. Thus, while preparative FF-IEF systems can fractionate large volumes of liquid with high recovery, the resulting fractions may have a net dilution of species, even for highly purified fractions. On an analytic scale, microfabricated FF-IEF devices may establish stable pH gradient without the need for additional pH buffers since the distance between electrodes is reduced. Such devices have been shown to effectively separate and concentrate species, such as fluorescent dyes, proteins and organelles, but often with lower resolution when compared to preparative systems.
Accordingly, improved devices and methods are needed.